An optional step is to treat the reaction with [http://www.neb.com/nebecomm/products/productM0303.asp RNase free DNaseI] to remove the template DNA.

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#Add 6 &mu;L DNaseI buffer

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#Add 3 &mu;L H<sub>2</sub>O

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#Add 1&mu;L DNaseI

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#Incubate 1 hr at 37&deg;C

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#Heat inactivate for 10 mins at 75&deg;C

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====Notes====

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*EDTA should be added to a final concentration of 5 mM to protect RNA from being degraded during enzyme inactivation. But EDTA can chelate magnesium which is needed for DNaseI activity. So the EDTA may need to be added just prior to inactivation.

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*DNase I is not active on DNA bound to proteins.

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*DTT which is typically included in ''in vitro'' transcription reactions can chelate zinc. Therefore, replace DTT with TCEP when the presence of zinc is necessary.

Following is the Knight:In vitro transcription protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder(see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|Vaishnavi]]

DNase treatment (optional)

An optional step is to treat the reaction with RNase free DNaseI to remove the template DNA.

Add 6 μL DNaseI buffer

Add 3 μL H2O

Add 1μL DNaseI

Incubate 1 hr at 37°C

Heat inactivate for 10 mins at 75°C

Notes

EDTA should be added to a final concentration of 5 mM to protect RNA from being degraded during enzyme inactivation. But EDTA can chelate magnesium which is needed for DNaseI activity. So the EDTA may need to be added just prior to inactivation.

DNase I is not active on DNA bound to proteins.

DTT which is typically included in in vitro transcription reactions can chelate zinc. Therefore, replace DTT with TCEP when the presence of zinc is necessary.

Analyze transcription products

Controls

There are a few controls that can help to ascertain that the assay is working properly.

Template without RNAP

To see what just the DNA template looks like when run on a gel.

Template with RNAP

To ensure that the linearized template is transcribed by RNAP.

Template with RNAP and the buffer that the repressor is resuspended in

To verify that any repression seen is not due to altered salt or pH conditions.

Notes

If using either plasmid DNA or DNA template has been linearized by restriction enzyme digestion, Ambion recommends a Proteinase K treatment followed by a phenol:chloroform extraction to eliminate all traces of RNase prior to subsequent reactions. This treatment is necessary because most plasmid DNA has been subjected to RNaseA during purification and restriction enzymes may be contaminated with RNases.

However, in the case of PCR generated templates, Ambion adds amplified DNA directly to the transcription reaction with no purification. 5 μl of a 100 μl PCR reaction (or about 0.05 - 0.2 μg of double-stranded DNA) is used as template. However, with shorter templates or low yields, the concentration of template in a 5 μl aliquot of the crude PCR reaction may be suboptimal. In that case, it may be desirable to concentrate the PCR product by alcohol precipitation. We do not generally find it necessary to phenol/chloroform extract the PCR reaction before precipitation, although in some cases it may be advisable to do so. [1]

References

BioCoder version

Following is the Knight:In vitro transcription protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder(see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi